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Administrative data

Description of key information

Weight of evidence: The oral sub-chronic NOAEL of the test item is deemed to be ca. 750 mg/kg bw/d (worst-case scenario), based on the available information from supporting analogue substances:

- Read-across from supporting substance (structural analogue or surrogate). Source: Key study. Method similar to OECD 408. The NOAEL of the analogue substance neohesperidin dihydrochalcone for oral sub-chronic toxicity in rats was found to be ca. 4000 mg/kg bw/d (5% in diet), and the NOEL ca. 750 mg/kg bw (1%). Based on the read-across approach, the NOAEL of the target substance is ca. 3987 mg/kg bw/d.

- Read-across from supporting substance (structural analogue or surrogate). Source: Study well documented, meets generally accepted scientific criteria (no guideline available). The NOAEL for the analogue substance hesperidin was set to 1% (ca. 750 mg) by weight per day in food. Based on the read-across approach, the NOAEL for oral toxicity of neohesperidin in rats can be set to ca. 750 mg/kg bw per day.

- Read-across from supporting substance (structural analogue or surrogate). Source: Method similar to OECD 408. The NOAEL of the analogue substance methyl hesperidin for oral sub-chronic toxicity in mice was found to be 5% in diet. Based on the read-across approach, the NOAEL of the target substance is 4.88% in diet.

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

Referenceopen allclose all

Endpoint:
sub-chronic toxicity: oral
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
1939
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
study well documented, meets generally accepted scientific principles, acceptable for assessment
Qualifier:
no guideline available
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity in Rodents)
Deviations:
yes
Remarks:
no analytical verification of doses, only 2 doses, only 6-8 males per group.
Principles of method if other than guideline:
As the study was performed in 1939, prior to any guideline being available, no guideline could be followed. However, basic scientific principles were followed incl. negative control (no treatment) and two different concentrations of test substance application.
GLP compliance:
no
Limit test:
no
Specific details on test material used for the study:
The sample used in this study had a yellowish color and decomposes at 251 °C and was supplied by the Los Angeles Laboratory of Fruit and Vegetable Chemistry of the Bureau of Chemistry aof Chemistry and Soils, U.S. Department of Agriculture.
- Name of test material (as cited in study report): hesperidin
- Molecular formula (if other than submission substance): C28H34O15
- Molecular weight (if other than submission substance): 610.5606
- Smiles notation (if other than submission s.): CC1C(O)C(O)C(O)C(OCC2C(O)C(O)C(O)C(Oc3cc4c(c(O)c3)C(=O)CC(c3ccc(OC)c(O)c3)O4)O2)O1
- InChl (if other than submission substance): InChI=1/C28H34O15/c1-10-21(32)23(34)25(36)27(40-10)39-9-19-22(33)24(35)26(37)28(43-19)41-12-6-14(30)20-15(31)8-17(42-18(20)7-12)11-3-4-16(38-2)13(29)5-11/h3-7,10,17,19,21-30,32-37H,8-9H2,1-2H3
- Structural formula attached as image file (if other than submission substance): see Fig. in attached report.
Species:
rat
Strain:
not specified
Remarks:
Albino
Sex:
male
Details on test animals and environmental conditions:
TEST ANIMALS
- Weight at study initiation: 50 g
- Diet ad libitum
- Water ad libitum
Route of administration:
oral: feed
Vehicle:
not specified
Details on oral exposure:
The test item was mixed with the stock diet in concentrations up to one percent by weight. The animals had free access to food and water at all times.
Analytical verification of doses or concentrations:
not specified
Duration of treatment / exposure:
200 days
Frequency of treatment:
continuous
Dose / conc.:
0.062 other: %
Remarks:
by weight (in food)
Dose / conc.:
1 other: %
Remarks:
by weight (in food)
No. of animals per sex per dose:
six to eight rats per dose level, male.
Control animals:
yes, concurrent no treatment
Details on study design:
Male albino rats averaging 50 grams body weight at the start of the feeding experiment were used, six to eight rats for each dosage level. The substance was mixed with the stock diet in concentrations up to one percent by weight.The animals had free access to food and water at all times.
Positive control:
no data
Observations and examinations performed and frequency:
DETAILED CLINICAL OBSERVATIONS: Yes

BODY WEIGHT: Yes

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: Not specified

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: No.
- After the rats had been on the diets two months, blood-sugar determinations were made on the controls and on those receiving the highest concentration of naringin.

CLINICAL CHEMISTRY: No

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: No

IMMUNOLOGY: No

OTHER: Organ weights.
Sacrifice and pathology:
GROSS PATHOLOGY: Yes. After 200 days all the rats were killed with ether and autopsied, and organs were weighed.

HISTOPATHOLOGY: Yes. Histological examination of paraffin sections stained with hematoxylin-eosin was performed on liver, heart, kidney, spleen, adrenal and testes.
Other examinations:
Blood-sugar determination after 2 months.
Statistics:
no data
Clinical signs:
no effects observed
Mortality:
no mortality observed
Body weight and weight changes:
effects observed, non-treatment-related
Description (incidence and severity):
During the last 50 days respiratory infection was observed throughout the laboratory colony, not related the test article affecting weight gain in this last period of experiment.
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
No signifficant differences in food intake between control and treated rats.
Food efficiency:
no effects observed
Description (incidence and severity):
Growth curves of negative controls and dose groups fully parallel.
Water consumption and compound intake (if drinking water study):
not specified
Ophthalmological findings:
not specified
Haematological findings:
no effects observed
Description (incidence and severity):
only blood sugar values were monitored.
Clinical biochemistry findings:
not specified
Urinalysis findings:
not specified
Behaviour (functional findings):
not specified
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
no effects observed
Description (incidence and severity):
The weights of the hearts, spleens, livers, adrenals, kidneys and testes were all within normal range and no macroscopical abnormalities were noted.
Gross pathological findings:
no effects observed
Description (incidence and severity):
During the last 50 days respiratory infection was observed throughout the laboratory colony, not related the test substance.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
no effects observed
Description (incidence and severity):
Histopathological examinations showed no significant morphological changes.
Histopathological findings: neoplastic:
not specified
Other effects:
no effects observed
Details on results:
At the end of 200 days all the rats were killed with ether and autopsied. The weights of the hearts, spleens, livers, adrenals kidneys, and testes were all within the normal range. All organs appeared normal range. All organs appeared normal macroscopically except the lungs of rats having respiratory infection. Histological examination of paraffin sections stained with hematoxylin-eosin showed no significant morphological changes in the livers, hearts, kidneys, spleens, adrenals and testes of rats receiving hesperidin.
Key result
Dose descriptor:
NOAEL
Effect level:
ca. 750 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male
Basis for effect level:
other: No effects were observed; 1% in diet applied to rats were converted to ca. 750 mg/kg/day calc ulated for rats with a mean body weight of ca. 250 g and ca. 20 g/day of food consumption.
Key result
Critical effects observed:
no
Conclusions:
No effects were observed in rats after oral administration of the test item at 1%. Therefore, the NOAEL can be set to 750 mg/kg bw per day (1%) in food for the target substance.
Executive summary:

A sub-chronic oral toxicity study was conducted with the analogue substance hesperidin (no guideline available, no analytical verification of doses). The study is well documented and meets generally accepted scientific principles. Six to eight healthy male albino rats per group were fed a standard diet containing the test item at doses of 0 (control), 0.0625 or 1% for 200 days. The continued feeding of the analogue substance to healthy albino rats in a standard diet for a period of 200 days, in concentrations as high as 1% of the diet by weight, gave no evidences of cumulative injury as judged by growth curves, weights of important viscera, and macroscopic and microscopic examination of these tissues. There was no difference between control rats and those receiving the test item, giving a negative result. Therefore, the NOAEL for the analogue substance can be set to 1% (ca. 750 mg) by weight per day in food. Based on the read-across approach, the NOAEL for oral toxicity of neohesperidin in rats can be set to 750 mg/kg bw per day.

Endpoint:
sub-chronic toxicity: oral
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
1993
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
study well documented, meets generally accepted scientific principles, acceptable for assessment
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity in Rodents)
Deviations:
yes
Remarks:
no analytical monitoring reported.
GLP compliance:
not specified
Limit test:
no
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: Hamari Pharmaceutical Ind., Ltd., Osaka, Japan, purity > 95.5%.

OTHER SPECIFICS:
- Name of test material (as cited in study report): methyl hesperidin
- Molecular formula (if other than submission substance): C29H36O15
- Molecular weight (if other than submission substance): 624,5871
- Smiles notation (if other than submission substance): CC1C(O)C(O)C(O)C(OCC2C(O)C(O)C(O)C(Oc3cc4c(c(O)c3)C(=O)CC(c3ccc(OC)c(OC)c3)O4)O2)O1
- InChl (if other than submission substance): InChI=1S/C29H36O15/c1-11-22(32)24(34)26(36)28(41-11)40-10-20-23(33)25(35)27(37)29(44-20)42-13-7-14(30)21-15(31)9-17(43-19(21)8-13)12-4-5-16(38-2)18(6-12)39-3/h4-8,11,17,20,22-30,32-37H,9-10H2,1-3H3/t11-,17-,20+,22-,23+,24+,25-,26+,27+,28+,29+/m0/s1
Species:
mouse
Strain:
B6C3F1
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Japan Inc., Kanagawa, Japan (120 mice; 60 males and 60 females).
- Age at study initiation: 6 weeks
- Housing: five animals were housed per plastic cage, on hardwood chips.
- Diet: powdered diet (Oriental MF, Oriental yeast Co., Ltd., Tokyo, Japan), ad libitum.
- Water: ad libitum.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 2
- Humidity (%): 50 ± 10
- Photoperiod (hrs dark / hrs light): 12h light/dark cycle.
Route of administration:
oral: feed
Vehicle:
not specified
Details on oral exposure:
DIET PREPARATION
- Mixing appropriate amounts with (Type of food): powdered diet (Oriental MF, Oriental yeast Co., Ltd., Tokyo, Japan) containing 0, 0.3, 0.6, 1.25, 2.5 and 5.0% methyl hesperidin (Hamari Pharmaceutical Ind., Ltd., Osaka, Japan, purity > 95.5%).
Analytical verification of doses or concentrations:
not specified
Duration of treatment / exposure:
13 weeks.
Frequency of treatment:
daily.
Dose / conc.:
0.3 other: % in diet.
Dose / conc.:
0.6 other: % in diet.
Dose / conc.:
1.25 other: % in diet.
Dose / conc.:
2.5 other: % in diet.
Dose / conc.:
5 other: % in diet.
No. of animals per sex per dose:
10 animals/sex/dose
Control animals:
yes
Details on study design:
- Dose selection rationale: not specified.
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Not specified
DETAILED CLINICAL OBSERVATIONS: Not specified

BODY WEIGHT: Yes
- Time schedule for examinations: weekly

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: Yes

FOOD EFFICIENCY:
- Body weight gain in kg/food consumption in kg per unit time X 100 calculated as time-weighted averages from the consumption and body weight gain data: Not specified

WATER CONSUMPTION AND COMPOUND INTAKE (not a drinking water study): Yes
- Time schedule for examinations: weekly.

OPHTHALMOSCOPIC EXAMINATION: Not specified

HAEMATOLOGY: Yes
- Time schedule for collection of blood: at week 13, blood samples were taken from the abdominal aorta.
- Anaesthetic used for blood collection: Yes (diethyl ether)
- Animals fasted: Yes
- How many animals:
- Parameters: erythrocyte and leukocyte counts, hematocrit and hemoglobin concentration using a Coulter Electronic Counter (Coulter Electronics, Inc., Hialeah, Fla.)

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: at week 13, blood samples were taken from the abdominal aorta.
- Animals fasted: Yes
- How many animals:
- Parameters: plasma analysis of glutamate-oxaloacetate transaminase (GOT), glutamate-pyruvate transaminase (GPT), alkaline phosphatase (ALP), total bilirubin (T-BIL), urea nitrogen (BUN), total cholesterol (T-CHO), glucose and total protein (TP), measured with an autoanalyzer (model ACA-8000 chemical analyzer, Olympus Optical Co., Tokyo, Japan).

URINALYSIS: Not specified
NEUROBEHAVIOURAL EXAMINATION: Not specified
IMMUNOLOGY: Not specified
Sacrifice and pathology:
GROSS PATHOLOGY: Yes
- At week 13, all mice were deprived of food overnight and then killed under diethyl ether anesthesia by exsanguination. A gross pathological examination was made on each animal during necropsy. The brain, heart, liver, spleen, kidneys, ovaries and testes were weighed before fixation along with other tissues in 10% buffered formalin.

HISTOPATHOLOGY: Yes
Histopathological examinations were performed on routinely prepared sections of heart, lymph nodes, bone marrow, thymus, spleen, pituitary, thyroid, adrenals, trachea, lungs, salivary glands, esophagus, stomach, small and large intestines, pancreas, liver, gall bladder, kidneys, urinary bladder, testes, prostate, seminal vesicles, mammary gland, ovaries, uterus, bone, skin, eyes, Harderian glands, brain, spinal cord, sciatic nerve and all gross lesions in the 0% and 5.0% groups, and heart, spleen, pancreas, liver, kidneys, lungs, brain and all gross lesions in the other groups.
Statistics:
The data were subjected to analysis of variance and differences between means were tested by Student’s t-test. The incidences of histopathologic lesions were analyzed by the two-sided Fisher’s exact probability test.
Clinical signs:
no effects observed
Mortality:
mortality observed, non-treatment-related
Description (incidence):
During the 13-week treatment period, deaths of 1 males in both the 1.25 and 3.0% methyl hesperidin groups occurred. The cause of death in each case showed no relationship to the treatment.
Body weight and weight changes:
effects observed, non-treatment-related
Description (incidence and severity):
Significant differences in body weight gains of males given 0.3% methyl hesperidin were observed at weeks 5 and 7 as compared with the control group. In the female groups, the mice receiving 0.3% and 0.6% methyl hesperidin demonstrated increased body weights at 7 weeks and thereafter. These changes were, however, not considered as effects of the treatment.
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
Average food and water consumption were similar in both sexes in all groups.
Food efficiency:
no effects observed
Description (incidence and severity):
Average food and water consumption were similar in both sexes in all groups.
Water consumption and compound intake (if drinking water study):
no effects observed
Description (incidence and severity):
Average food and water consumption were similar in both sexes in all groups.
Ophthalmological findings:
not specified
Haematological findings:
no effects observed
Description (incidence and severity):
Hematological parameters showed no significant differences among the groups in either males or females.
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
In blood chemistry, glucose levels were significantly lower in female mice given 2.5 and 5.0% methyl hesperidin than in the controls. However, there was no clear dose-dependence and no chemical effects on any of the other clinical chemistry parameters at any dosage level were noted.
Urinalysis findings:
not specified
Behaviour (functional findings):
not specified
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, non-treatment-related
Description (incidence and severity):
Significant differences in the absolute and relative weights of the heart and kidneys were sporadically observed, but this was not dose dependent and there were no related histopathological findings.
Gross pathological findings:
effects observed, non-treatment-related
Description (incidence and severity):
On gross pathological examination, injury to the penis was noted in one or two male mice in the 0.3, 0.6 and 2.5% dose groups. However, it was considered a consequence of dysuria in fighting mice.
Neuropathological findings:
not specified
Histopathological findings: non-neoplastic:
effects observed, non-treatment-related
Description (incidence and severity):
Development of nephritis in the 0.3, 0.6 and 2.5% dose groups. However, it was considered a consequence of dysuria in fighting mice. In the other organs, no histopathological findings suggestive of toxicity were observed in any group.
Histopathological findings: neoplastic:
not specified
Other effects:
no effects observed
Key result
Dose descriptor:
NOAEL
Effect level:
5 other: % in diet
Based on:
test mat.
Sex:
male/female
Basis for effect level:
clinical biochemistry
Key result
Critical effects observed:
no

Table 1. Hematology data for mice fed methyl hesperidin for 13 weeks.

Conc. (%)

No. of mice

Erythrocytes

(x 1000)

Leukocytes

(x 1000)

Hemoglobin

(g/dl)

Hematocrit

(%)

Females

0

10

1012 ± 46

73.4 ± 29.3

17.0 ± 1.1

47.5 ± 2.0

0.3

10

1013 ± 99

60.7 ± 9.9

17.1 ± 1.6

48.0 ± 5.2

0.6

10

1032 ± 56

65.7 ± 18.1

17.3 ± 1.5

48.4 ± 2.6

1.25

10

1048 ± 76

80.1 ± 32.5

17.3 ± 1.7

48.6 ± 3.8

2.5

10

1023 ± 90

91.5 ± 42.4

17.1 ± 1.5

47.8 ± 3.6

5.0

10

1015 ± 49

64.6 ± 18.3

17.2 ± 1.7

47.5 ± 2.5

Males

0

10

1023 ± 101

156.5 ± 175.7

17.3 ± 1.5

48.0 ± 4.3

0.3

9

1005 ± 105

113.7 ± 77.2

16.8 ± 2.2

45.5 ± 5.1

0.6

10

979 ± 146

188.7 ± 175.7

16.5 ± 2.4

46.1 ± 5.6

1.25

9

1037 ± 74

110.4 ± 47.3

17.2 ± 2.0

48.9 ± 3.8

2.5

10

1031 ± 90

110.3 ± 70.1

17.4 ± 1.6

48.1 ± 3.9

5.0

10

1030 ± 47

88.0 ± 29.0

17.5 ± 1.8

48.4 ± 2.8

 

Table 2. Clinical chemistry data for mice fed methyl hesperidin for 13 weeks.

Conc.

(%)

No. of

mice

GOT

(KU)

GPT

(KU)

ALP

(KAU)

T.B±L

(mg/dl)

BUN

(mg/dl)

T.CHO

(mg/dl)

Glucose

(mg/dl)

TP

(mg/dl)

Females

0

10

106 ± 47.2

24.8 ± 12.6

8.3 ± 1.0

0.20 ± 0.11

28.3 ± 3.5c

79.2 3 ± 6.7c

190.5 ± 26.2c

5.2 ± 0.1c

0.3

10

95.2 ± 32.5

31.7 ± 24.6

8.2 ± 1.1

0.20 ± 0.14

23.0 ± 2.5b**

82.0 3 ± 13.9c

182.4 3 ± 17.6d

5.3 ± 0.2b

0.6

10

81.9 ± 15.0

19.7 ± 14.1

8.1 ± 0.8

0.20 ± 0.13

26.7 ± 3.5

81.1 3 ± 6.1

174.1 ± 17.8b

5.2 ± 0.3

1.25

10

102.3 ± 22.2

35.3 ± 23.3

9.1 ± 0.6

0.20 ± 0.15

27.0 ± 7.4d

86.6 3 ± 5.5c,*

171.7 ± 11.8c

5.0 ± 0.6c

2.5

10

96.9 ± 16.3

22.8 ± 7.4

8.3 ± 0.6

0.20 ± 0.14

28.8 ± 6.3

79.2 ± 12.6c

157.0 ± 21.0°*

5.1 ± 0.2c

5.0

10

109.3 ± 34.3

38.2 ± 19.5

8.8 ± 0.8

0.20 ± 0.04

28.8 ± 6.8

88.5 ± 7.2*

157.7 ± 24.2*

5.1 ± 0.2

Males

0

10

105.2 ± 22.8

32.0 ± 18.2

6.2 ± 0.9

0.23 ± 0.25

33.6 ± 3.8

108.7 ± 11.8

157.7 ± 21.1b

5.5 ± 0.3

0.3

9

89.0 ± 18.0

37.7 ± 20.5

6.4 ± 1.4

0.15 ± 0.07

30.7 ± 4.3

109.2 ± 8.3b

160.7 ± 31.6b

5.3 ± 0.2b

0.6

10

93.9 ± 28.6

24.9 ± 11.2

5.3 ± 1.3

0.23 ± 0.20

29.2 ± 7.4

110.0 ± 11.3

142.2 ± 28.8

5.3 ± 0.2

1.25

9

80.8 ± 10.4*

23.8 ± 4.5

6.1 ± 0.9

0.16 ± 0.13

31.1 ± 6.8

106.7 ± 8.8

146.7 ± 25.7

5.4 ± 0.2

2.5

10

93.9 ± 30.1

26.1 ± 14.8

5.8 ± 1.1

0.20 ± 0.15

29.6 ± 8.9b

109.0 ± 8.7b

182.0 ± 43.4b

5.3 ± 0.3b

5.0

10

104.2 ± 35.1

42.1 ± 27.7

6.2 ± 2.0

0.13 ± 0.05

32.6 ± 8.9b

113.6 ± 13.7b

164.0 ± 32.1d

5.4 ± 0.3b

*, **Significantly different from control group value at P < 0.05, 0.01, respectively.

a) Data are mean ± SD values.

b) Sample volume from one animal was insufficient for complete analysis.

c) Sample volume from two animals was insufficient for complete analysis.

d) Sample volume from three animals was insufficient for complete analysis.

Table 3. Mean final body and absolute organ weights in mice fed methyl hesperidin for 13 weeks.

Cone.

(%)

No.

of mice

Final

body weight

(g)

Absolute organ weights (g)

brain

heart

liver

spleen

kidneys

ovaries/testes

Female

0

10

24.6 ± 1.0a

0.49 ± 0.02

0.13 ±0.01

1.02 ± 0.07

0.09 ± 0.01

0.33 ± 0.03

0.021 ± 0.005

0.3

10

25.6 ± 1.3

0.49 ± 0.02

0.13 ± 0.01

1.07 ± 0.10

0.09 ± 0.02

0.35 ± 0.02

0.023 ± 0.005

0.6

10

25.5 ± 1.4

0.49 ± 0.02

0.14 ± 0.01

1.07 ± 0.07

0.09 ±0.01

0.35 ± 0.02

0.022 ± 0.005

1.25

10

25.2 ± 1.6

0.50 ± 0.01

0.13 ± 0.02

1.08 ± 0.10

0.09 ± 0.01

0.36 ± 0.02*

0.023 ± 0.004

2.5

10

24.4 ± 0.9

0.50 ± 0.03

0.13 ±0.01

1.04 ± 0.06

0.09 ± 0.01

0.36 ± 0.02*

0.035 ± 0.035

5.0

10

24.4 ± 1.4

0.49 ± 0.02

0.12 ±0.01

1.07 ± 0.07

0.09 ± 0.01

0.35 ± 0.02

0.022 ± 0.005

Male

0

10

30.0 ± 2.3

0.47 ± 0.01

0.16 ± 0.01

1.27 ± 0.17

0.11 ± 0.03

0.49 ± 0.05

0.23 ± 0.01

0.3

9

29.8 ± 3.6

0.46 ± 0.02

0.15 ± 0.02

1.22 ± 0.16

0.10 ± 0.03

0.47 ± 0.05

0.23 ± 0.02

0.6

10

30.1 ± 1.8

0.47 ± 0.02

0.15 ± 0.01

1.29 ± 0.12

0.14 ± 0.05

0.47 ± 0.04

0.23 ± 0.01

1.25

9

29.0 ± 1.3

0.47 ± 0.01

0.14 ± 0.01*

1.19 ± 0.09

0.11 ± 0.03

0.48 ± 0.04

0.23 ± 0.01

2.5

10

29.4 ± 2.1

0.46 ± 0.01

0.16 ± 0.02

1.26 ± 0.13

0.11 ± 0.04

0.46 ± 0.04

0.23 ± 0.02

5.0

10

29.0 ± 2.3

0.47 ± 0.01

0.15 ± 0.01

1.22 ± 0.16

0.09 ± 0.02

0.45 ± 0.04

0.23 ± 0.02

Data are mean ± SD values.

*,** Significantly different from control group value at P < 0.05.

Table 4. Mean relative organ weights in mice fed methyl hesperidin for 13 weeks.

Conc.

(%)

No.

of mice

Relative organ weights (%)

brain

heart

liver

spleen

kidneys

ovaries/testes

Female

0

10

1.98 ± 0.11a

0.53 I 0.05

4.16 ±0.20

0.36 ± 0.05

1.35 ± 0.06

0.086 ± 0.020

0.3

10

1.90 ± 0.11

0.50 ± 0.06

4.20 ± 0.23

0.35 ± 0.04

1.38 ± 0.08

0.091 ± 0.020

0.6

10

1.92 ± 0.14

0.55 ± 0.06

4.20 ± 0.32

0.35 ± 0.04

1.39 ± 0.10

0.087 ± 0.019

1.25

10

1.98 ± 0.14

0.52 ± 0.09

4.30 ± 0.29

0.36 ± 0.03

1.43 ± 0.09*

0.092 ± 0.019

2.5

10

2.05 ± 0.12

0.52 ± 0.06

4.27 ± 0.20

0.37 ± 0.04

1.46 ± 0.04**

0.142 ± 0.147

5.0

10

1.99 ± 0.11

0.49 ± 0.04

4.37 ± 0.24

0.39 ± 0.04

1.42 ± 0.08*

0.091 ± 0.020

Male

0

10

1.57 ± 0.14

0.53 ± 0.03

4.22 ± 0.31

0.37 ± 0.08

1.62 ± 0.08

0.78 ± 0.05

0.3

9

1.58 ± 0.17

0.50 ± 0.07

4.11 ± 0.44

0.34 ± 0.12

1.59 ± 0.12

0.78 ± 0.07

0.6

10

1.57 ± 0.08

0.50 ± 0.02*

4.27 ± 0.36

0.47 ± 0.17

1.55 ± 0.12

0.75 ± 0.04

1.25

9

1.61 ± 0.06

0.50 ± 0.04

4.10 ± 0.22

0.37 ± 0.09

1.67 ± 0.12

0.80 ± 0.04

2.5

10

1.58 ± 0.11

0.53 ± 0.04

4.29 ± 0.32

0.37 ± 0.14

1.56 ± 0.10

0.79 ± 0.05

5.0

10

1.63 ± 0.13

0.53 ± 0.06

4.20 ± 0.38

0.31 ± 0.06

1.54 ± 0.07*

0.81 ± 0.07

a) Data are mean ± SD values.

*,**Significantly different from control group value at P < 0.05, 0.01, respectively.

Conclusions:
The test item has a sub-chronic oral NOAEL of 5.0% in the diet in B6C3F1 mice.
Executive summary:

A subchronic toxicity study was conducted for methyl hesperidin by a method similar to OECD 408. One hundred and twenty B6C3F1 mice (60 males and 60 females) were fed powdered diet containing 0 (control), 0.3, 0.6, 1.25, 2.5 and 5.0% test substance for 13 weeks. No significant changes were revealed in any of the measured parameters, except for the clinical chemistry: females fed 2.5% and 5.0% test substance demonstrated significant decreases in glucose level. However, the lack of any clear dose-relation or adverse effects on body weight, organ weight, gross pathological and histopathological appearance indicated an absence of toxicity. Thus, it can be concluded that methyl hesperidin exerts no toxic influence in mice of either sex at levels up to 5.0% in the diet. Based on the study results, the NOAEL for the test item is set at 5.0% in diet.

Endpoint:
sub-chronic toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
1990
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
comparable to guideline study with acceptable restrictions
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity in Rodents)
Deviations:
not specified
GLP compliance:
not specified
Limit test:
no
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: supplied by Zoster S.A. (Raiguero, S/N, Zeneta-Murcia, Spain), lot no.1999
- Purity: > 99.5%

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: The sample was stored in sealed plastic bags in a cool and dry place.
- Stability under test conditions: NHDC was found to be stable in the diet under the experimental conditions applied.

OTHER SPECIFICS:
- Name of test material (as cited in study report): Neohesperidin dihydrochalcone (NHDC).
- Appearance: white crystalline, finely ground, non-hygroscopic powder.
- Molecular formula (if other than submission substance): C28H34O15
- Molecular weight (if other than submission substance): 610.5606
- Smiles notation (if other than submission s.): CC1C(O)C(O)C(O)C(OCC2C(O)C(O)C(O)C(Oc3cc4c(c(O)c3)C(=O)CC(c3ccc(OC)c(O)c3)O4)O2)O1
- InChl (if other than submission substance): InChI=1/C28H36O15/c1-11-21(34)23(36)25(38)27(40-11)43-26-24(37)22(35)19(10-29)42-28(26)41-13-8-16(32)20(17(33)9-13)14(30)5-3-12-4-6-18(39-2)15(31)7-12/h4,6-9,11,19,21-29,31-38H,3,5,10H2,1-2H3
Species:
rat
Strain:
Wistar
Remarks:
(Cpb: WU; Wistar random)
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: TNO Central Institute for the Breeding of Laboratory Animals, Zeist, The Netherlands.
- Weight at study initiation: males weighed around 84 g and females around 79 g.
- Housing: The rats were housed in groups of five of the same sex and treatment group in suspended stainless-steel wire-screen cages.
- Diet: cereal-based, open-formula diet (TNO-C1VO Toxicology and Nutrition Institute, the Netherlands). Food was provided ad libitum.
- Water: tap water, ad libitum.
- Acclimation period: 7 days

DETAILS OF FOOD AND WATER QUALITY: The Institute's basal diet is analysed for both nutrients and contaminants twice a year. Typical results of analysis for calcium, phosphorus and magnesium are: Ca, 0.7-0.9%; P, about 0.6%; Mg, about 0.17%.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 2ºC
- Humidity (%): 40-80%
- Air changes (per hr): 10
- Photoperiod (hrs dark / hrs light): 12-hr light/dark cycle.
Route of administration:
oral: feed
Vehicle:
unchanged (no vehicle)
Details on oral exposure:
DIET PREPARATION
- Rate of preparation of diet (frequency): Fresh batches of the test diets were prepared five times in the course of the study.
- Mixing appropriate amounts with (Type of food): NHDC was mixed into the Institute's cereal-based, open-formula diet, the composition (%) of which was as follows: soybean oil meal 11; fish meal 7; meat and bone scraps 4; whole ground wheat 36; whole ground maize 29.7, grass meal 3; whey powder 2; defatted bone meal 0.4; soybean oil 3; trace elements in salt 0.5; brewers' yeast 3; vitamin B preparation 0.1; vitamin ADEK mixture 0.3. Typical results of analysis for calcium, phosphorus and magnesium are: Ca, 0.7-0.9%; P, about 0.6%; Mg about 0.17%.
- Storage temperature of food: The diets were stored at about 15ºC.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
- Diet samples were analysed for NHDC content by HPLC, according to Fisher (1977), using an M&N 25cm x 4.6mm i.d. reverse-phase Poligosil 60-5-CI8 column. The adequacy of the mixing procedure was investigated by analysing five samples of each test diet from the first batch. The stability of NHDC in this batch of diets was examined after storage for 4 wk at room temperature. In addition, the NHDC content of the test diets was analysed monthly.
- Throughout the study, the actual levels of NHDC in the low-, intermediate- and high-dose diet were 95%, 97-99% and 94-105% of the intended levels, respectively. The quintuplicate analyses of the first batch of each of the NHDC-containing diets showed coefficients of variation of less than 5%, indicating satisfactory homogeneity.
Duration of treatment / exposure:
13 consecutive weeks.
Frequency of treatment:
Daily.
Dose / conc.:
0 mg/kg bw/day (nominal)
Remarks:
Control.
Dose / conc.:
150 mg/kg bw/day (nominal)
Remarks:
0.2 % (w/w) in diet, ca. 150 mg/kg (males) - 166 mg/kg (females).
Dose / conc.:
750 mg/kg bw/day (nominal)
Remarks:
1% (w/w) in diet, ca. 757 mg/kg (males) - 848 mg/kg (females).
Dose / conc.:
4 000 mg/kg bw/day (nominal)
Remarks:
5 % (w/w) in diet, ca. 4011 mg/kg (males) - 4334 mg/kg (females).
No. of animals per sex per dose:
20 rats/sex/group.
Control animals:
yes, plain diet
Details on study design:
- Dose selection rationale: Groups of 20 male and 20 female rats were fed diets containing 0, 0.2, 1.0 or 5.0% NHDC for 13 consecutive weeks. No further details.
- Owing to the normal decrease in food intake per unit body weight with increasing age of rats, the intake of NHDC per kg body weight became lower in the course of the study. The overall actual intakes of NHDC were roughly 150, 750 and 4000 mg/kg body weight/day for the low-, intermediate- and high-dose groups, respectively.
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: daily.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: The rats were observed daily for external signs of toxicity.

BODY WEIGHT: Yes
- Time schedule for examinations: The rats were weighed weekly

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: Yes. Food intake was measured over 1-wk periods.

FOOD EFFICIENCY:
- Body weight gain in kg/food consumption in kg per unit time X 100 calculated as time-weighted averages from the consumption and body weight gain data: Yes

WATER CONSUMPTION AND COMPOUND INTAKE (not a drinking water study): Yes
- Time schedule for examinations: water intake was recorded in wk 7 and 12.

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: before the start of the study and at wk 13.
- Dose groups that were examined: control group and high-dose group.

HAEMATOLOGY: Yes
- Time schedule for collection of blood: early in week 13.
- Anaesthetic used for blood collection: No
- Animals fasted: Not specified.
- How many animals: 10 rats/sex/group
- Parameters examined: haemoglobin concentration, packed cell volume, and counts of thrombocytes and red and white blood cells (including a differential white cell count).

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: in wk 13.
- Animals fasted: Yes
- How many animals: 10 rats/sex/group
- Parameters examined: glucose (Boehringer Glucoquant No. 245-178; Boehringer Mannheim GmbH Mannheim, FRG); alkaline phosphatase, aspartate aminotransferase, alanine aminotransferase, r-glutamyltransferase, total protein, albumin, total bilirubin, urea, cholesterol, creatinine and calcium (Cobas-Bio Centrifugal Analyzer), ornithine-carbamyl transferase (Spectro-fotometric), inorganic phosphate (Boehringer Kit No. 124-974, Boehringer Mannheim GmbH), chloride (Chloro Counter, Marius), and sodium and potassium (Electrolyte-2-Analyzer, Beckman).

URINALYSIS: Yes
- Time schedule for collection of urine: urine was collected from ten rats/sex/group during the last 16 hr of a 24-hr period of deprivation of food and water in wk 13.
- Metabolism cages used for collection of urine: Not specified
- Animals fasted: Yes
- Parameters examined: volume (calibrated tubes), density (Bellingham & Stanley refractometer), for protein, glucose, occult blood, ketones, bilirubin and urobilinogen (pooled samples; Combur test strips, Boehringer Mannheim GmbH), and microscopy of the sediment (pooled samples).

OTHER: In week 13, pH was determined in urine samples collected over 3 hr early in the morning from ten non-fasted rats/sex/group (Philips P.W. 9410 pH meter).

NEUROBEHAVIOURAL EXAMINATION: No
IMMUNOLOGY: No
Sacrifice and pathology:
GROSS PATHOLOGY: Yes
- Early in wk 14, the rats were killed by exsanguination from the abdominal aorta, under light ether anaesthesia, and subjected to a thorough post-mortem examination. Any abnormalities were recorded and the adrenals, brain, caecum (with and without contents), gonads, heart, kidneys, liver, spleen, thymus and thyroid (with parathyroids) were weighed. Samples of these organs and of the aorta, axillary lymph nodes, colon, duodenum, epididymides, eyes, ileum, jejunum, lungs, mammary glands (both sexes), mesenteric lymph nodes, oesophagus, pancreas, pituitary, prostate, rectum, sciatic nerve, skeletal muscle, spinal cord, sternum, stomach (glandular and non-glandular), salivary glands (parotid, sublingual and submaxillary), trachea, urinary bladder and uterus (with cervix) were preserved in 10% phosphate buffered formaldehyde.

HISTOPATHOLOGY: Yes
- Wax-embedded 5-11 m sections of the preserved tissues from all animals in the control and high-dose group were stained with haematoxylin and eosin and examined microscopically. One female of the intermediate-dose group that died intercurrently was subjected to histopathological examination as well.
Statistics:
Data on body weights were evaluated by one-way analysis of covariance, followed by Dunnett's multiple-comparison tests. The laboratory determinations and organ weights were evaluated by one-way analysis of variance, followed by Dunnett's multiple-comparison tests, except for the differential white blood cell counts and urinary pH values which were analysed by the Mann Whitney U test. Data on food and liquid intake was evaluated by analysis of variance, followed by least significant difference tests (experimental unit: the cage). Fisher's exact probability test (two sided) was used for results of ophthalmoscopy and for pathology data.
Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
No treatment-related changes were observed in the low- and intermediate-dose groups. In the high-dose group, both males and females showed soft stools in wk 2 and 3.
Mortality:
mortality observed, non-treatment-related
Description (incidence):
One female rat in intermediate dose group was found dead at day 30 but, as no additional mortality was found in any dose groups and macroscopic and microscopic examination of the animal found dead revealed renal failure not seen in any other dose groups, this death is considered a fortuitous finding.
Body weight and weight changes:
effects observed, non-treatment-related
Description (incidence and severity):
The low and medium dose groups showed no effects. At the high-dose level, growth was depressed in males throughout the study and in females in the first 2 weeks and, accordingly, food intake slightly decreased.
Growth retardation may have been caused by interference by NHDC and/or its metabolites in the absorption of nutrients. However, the poor growth was accompanied by a transient reduction in food intake and food efficiency. Hence, the slight growth retardation is more likely due to the reduced food intake observed in the first 2 wk.
Food consumption and compound intake (if feeding study):
effects observed, non-treatment-related
Description (incidence and severity):
The low and medium dose group showed no effects. At the high-dose level in males throughout the study and in females in the first 2 weeks food intake was slightly decreased. The high level of NHDC in the diet probably decreased acceptability, causing poor food consumption until the rats had adapted to the taste of the product.
Food efficiency:
effects observed, non-treatment-related
Description (incidence and severity):
The low and medium dose group showed no effects. At the high-dose level a transient decrease in food-conversion efficiency was observed. The reduced food efficiency is in line with the assumption that NHDC decreased acceptability, since when food intake is restricted, a greater percentage is required for maintenance and a reduced percentage is
available for growth.
Water consumption and compound intake (if drinking water study):
no effects observed
Description (incidence and severity):
Water consumption was not significantly affected by the administration of NHDC at any dose level.
Ophthalmological findings:
no effects observed
Description (incidence and severity):
Ophthalmoscopic examinations did not reveal any treatment related effects.
Haematological findings:
no effects observed
Description (incidence and severity):
There were no significant changes in haematology among the groups.
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
Biochemical changes that might have been due to treatment, were observed in the high-dose group only: in females, plasma alkaline phosphatase activity and bilirubin concentration were increased; total plasma protein concentration was decreased in males; plasma urea concentration was relatively low in both sexes.
Three of the changes in clinical chemistry variables (viz increased plasma alkaline phosphatase activity, low plasma urea concentration and decreased urinary pH) were considered to be related to the metabolic fate of the test item.
Urinalysis findings:
effects observed, treatment-related
Description (incidence and severity):
Urinary pH was decreased in both sexes of high-dose group.
Behaviour (functional findings):
no effects observed
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
- The organ-weight data showed marked enlargement of the caecum in the high-dose group in both sexes. Slight, though statistically significant increases in caecal weight occured also in males of the low- and intermediate-dose group but there was no dose-response relationship.
- The relative weights of the brain and testicles were increased in males of the high-dose group. The mean absolute weights of these organs were, however, very similar to those of the controls. This change is not ascribed to a direct effect of the test item. The other organ weights did not show treatment-related changes in either sex.
Gross pathological findings:
no effects observed
Description (incidence and severity):
Gross examination at autopsy did not reveal treatment-related changes in any of the experimental groups.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
no effects observed
Description (incidence and severity):
Microscopic examination of the organs and tissues from all rats of the control and high-dose group did not reveal any treatment-related changes.
Histopathological findings: neoplastic:
not specified
Other effects:
no effects observed
Details on results:
The present study has shown that the administration of the test item to rats at dietary levels of up to 5% for 13 wk, results only at the high-dose level in treatment-related changes, namely, marginal effects on body weight and food consumption, distinct caecal enlargement and slight changes in some of the clinical chemistry variables. These changes were judged to be of little, if any, toxicological significance.
Key result
Dose descriptor:
NOEL
Effect level:
ca. 750 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
clinical biochemistry
organ weights and organ / body weight ratios
Key result
Dose descriptor:
NOAEL
Effect level:
ca. 4 000 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
clinical biochemistry
organ weights and organ / body weight ratios
Key result
Critical effects observed:
no

Table 1. Body weights, food consumption and test item intakes of rats fed diets containing 0-5% NHDC for 13 wk.

Dose

(% in diet)

Body weight (g) at week

Food consumption (g/rat/wk)

Mean intake**

(mg/kg bw/d)

Males

0

1

2

3

4

8

13

1

2

3

4

8

12

0

84 ± 0.9

125 ± 1.4

166 ± 2.4

214 ± 3.7

255 ± 4.4

343 ± 7.0

392 ± 8.3

105

128

135

149

148

147

0

0.2

84 ± 0.9

125 ± 1.7

166 ± 2.8

212 ± 3.9

254 ± 4.4

341 ± 9.1

391 ± 11.0

106

128

133

148

142

146

150

1.0

84 ± 1.1

125 ± 1.8

164 ± 2.6

210 ± 3.8

249 ± 4.1

338 ± 5.6

387 ± 6.5

106

126

134

147

146

146

757

5.0

84 ± 0.9

125 ± 2.2*

146 ± 3.3*

192 ± 3.4*

232 ± 3.8*

310 ± 6.5*

355 ± 7.4

93*

111*

132

148

142

146

4011

Females

 

0

79 ± 0.9

110 ± 1.2

133 ± 1.6

156 ± 2.4

172 ± 2.4

210 ± 4.3

230 ± 5.1

92

100

103

106

111

109

0

0.2

79 ± 0.8

110 ± 1.1

135 ± 1.6

159 ± 2.3

174 ± 2.7

213 ± 3.7

234 ± 4.2

91

101

100

105

112

109

166

1.0

79 ± 1.2

109 ± 1.5

132 ± 2.0

154 ± 2.8

169 ± 2.8

206 ± 4.6

232 ± 4.5

91

102

98

106

111

111

848

5.0

79 ± 0.9

104 ± 1.6*

126 ± 2.1*

153 ± 2.7

170 ± 2.9

206 ± 4.1

227 ± 4.2

87

100

102

108

111

113

4334

**Overall means of weekly calculations from data on body weight, food intake and nominal levels of NHDC.

Body-weight values are means ± SEM for groups of 20 rats. Food consumption values are the means for four cages of five animals. Although growth and food consumption were recorded weekly, only values obtained during the first 4 weeks and subsequendly monthly are presented. The values marked with * differ significantly (body weight; Dunnett’s test, and food consumption; least significant difference test) from the control value (*P < 0.01).

Table 2. Plasma biochemical and urinary parameters of rats fed diets containing 0-5% NHDC for 13 wk.

Parameter

% NHDC in diet

0

0.2

1.0

5.0

MALES

Plasma

Alkaline phosphatase (U/L)

144.9 ± 7.0

141.0 ± 5.8

163.9 ± 7.2

150.3 ± 4.0

Total protein (g/L)

63.4 ± 0.9

61.3 ± 0.4

61.7 ± 0.5

59.6 ± 0.6**

Bilirubin (μmol/L)

0.30 ± 0.13

0.20 ± 0.08

0.02 ± 0.01

0.61 ± 0.24

Urea (mmol/L)

7.08 ± 0.29

7.68 ± 0.55

7.76 ± 0.34

6.50 ± 0.14

Urine

pH

8.0

7.5

7.8

7.2**

FEMALES

Plasma

Alkaline phosphatase (U/L)

129.1 ± 4.7

124.8 ± 8.9

142.8 ± 5.9

154.4 ± 5.1*

Total protein (g/L)

61.7 ± 0.6

61.0 ± 0.5

60.9 ± 0.8

61.6 ± 0.7

Bilirubin (μmol/L)

0.32 ± 0.13

0.25 ± 0.09

0.62 ± 0.10

1.04 ± 0.17**

Urea (mmol/L)

8.06 ± 0.75

8.52 ± 0.71

7.78 ± 0.50

6.34 ± 0.33

Urine

pH

8.0

7.9

7.9

7.3**

Values are means ± SEM for groups of ten rats; those marked with asterisks differ significantly [Dunnett’s test or Mann-Whitney U-test (for pH values)] from the corresponding control value (*P < 0.05; **P < 0.01). No other blood, plasma or urinary analysis values differed significantly from the corresponding control value.

Table 3. Relative organ weights of rats fed diets containing 0-5% NHDC for 13 wk.

Organ

Relative organ weights (g/kg bw)

0

0.2

1.0

5.0

MALES

Adrenals

0.132 ± 0.003

0.140 ± 0.004

0.145 ± 0.004

0.142 ± 0.005

Brain

5.13 ± 0.12

5.04 ± 0.14

5.12 ± 0.07

5.55 ± 0.13*

Caecum (full)

11.6 ± 0.6

14.8 ± 0.6**

14.5 ± 0.6**

20.4 ± 0.9**

Caecum (empty)

2.3 ± 0.1

2.8 ± 0.1*

2.6 ± 0.1

3.5 ± 0.2**

Heart

3.21 ± 0.06

3.32 ± 0.07

3.18 ± 0.06

3.28 ± 0.06

Kidneys

6.29 ± 0.13

6.31 ± 0.11

6.28 ± 0.12

6.33 ± 0.08

Liver

36.0 ± 0.6

36.2 ± 0.7

35.4 ± 0.5

34.1 ± 0.5

Spleen

1.60 ± 0.04

1.71 ± 0.05

1.64 ± 0.05

1.60 ± 0.04

Testes

8.55 ± 0.20

8.54 ± 0.28

8.65 ± 0.23

9.49 ± 0.23*

Thymus

0.92 ± 0.05

0.99 ± 0.05

0.85 ± 0.04

0.88 ± 0.04

Thyroid

0.063 ± 0.003

0.066 ± 0.003

0.062 ± 0.004

0.067 ± 0.003

FEMALES

Adrenals

0.286 ± 0.010

0.285 ± 0.010

0.285 ± 0.09

0.309 ± 0.009

Brain

7.90 ± 0.18

7.71 ± 0.12

7.78 ± 0.15

7.94 ± 0.13

Caecum (full)

15.5 ± 0.6

14.7 ± 0.7

16.6 ± 0.7

22.5 ± 0.8**

Caecum (empty)

3.3 ± 0.2

3.3 ± 0.2

3.5 ± 0.1

4.6 ± 0.2**

Heart

3.85 ± 0.07

3.85 ± 0.06

3.87 ± 0.09

3.90 ± 0.07

Kidneys

6.71 ± 0.09

6.58 ± 0.13

6.62 ± 0.10

6.85 ± 0.09

Liver

33.4 ± 0.3

33.3 ± 0.5

33.0 ± 0.5

33.9 ± 0.5

Spleen

1.86 ± 0.05

1.95 ± 0.05

1.84 ± 0.04

1.95 ± 0.04

Ovaries

0.363 ± 0.013

0.372 ± 0.014

0.377 ± 0.013

0.347 ± 0.010

Thymus

1.27 ± 0.05

1.31 ± 0.04

1.29 ± 0.04

1.36 ± 0.05

Thyroid

0.092 ± 0.003

0.082 ± 0.04

0.089 ± 0.004

0.094 ± 0.003

Values are means ± SEM for groups of 20 rats. Those marked with asterisks differ significantly (Dunnett’s test) from the corresponding control value (*P < 0.05; **P < 0.01).

Table 4. Major histopathological changes in rats fed NHDC for 13 wk.

Organ and lesion

Dose(%):

No. of rats affected

Males

Females

0

5.0

0

5.0

Adrenals

- Cortical vacuolation

7

4

0

0

Epididymis

- Reduced spermatogenesis

1

0

 

 

Heart

- Cartilaginous metaplasia

0

1

0

1

Kidneys

- Nephrosis

13

11

3

5

- Calcareous deposits

in the cortico-medullary layer

0

1

14

11

- Calcareous deposits in pelvis

0

0

4

0

- Urothelial hyperplasia in pelvis

0

0

3

0

- Hydronephrosis

0

1

0

1

Large intestines

- Parasites

2

1

1

3

Liver

- Aggregates of RES cells

3

4

5

5

Lung

- Perivascular lymphocytic infiltrates

1

1

9

3

Pituitary

- Cysts

1

1

1

2

- Tubular hyperplasia in pars neuralis

0

0

0

1

Spleen

- Hematopoiesis

0

1

0

1

Stomach

- Fundic glandular dilatation

0

0

3

0

Sub-lingual salivary glands

- Demi-lune cell proliferation

0

0

0

2

Testes

- Atrophy

1

0

 

 

- Tubular calcareus deposits

2

0

 

 

Urinary bladder

- Urothelial hyperplasia

0

0

0

1

Uterus

- Polyp

 

 

0

1

- Endometritis

 

 

0

2

The tissues of 20 animals were examined microscopically in all cases except that the pituitaries of only 19 control males were examined.

Conclusions:
The test item has a sub-chronic oral NOAEL of ca. 4000 mg/kg bw/d (5% in diet) in rats, and a NOEL of ca. 750 mg/kg bw/d (1% in diet), based on clinical findings and biochemistry.
Executive summary:

A study on the sub-chronic toxicity of the test item was performed by a method similar to OECD 408, without GLP. The test item was administered to groups of 20 male and 20 female Wistar rats at dietary levels of 0 (control), 0.2, 1.0 and 5.0% for 91 days. No treatment-related ophthalmoscopical, haematological or histopathological effects were observed. In the high-dose group, a marked caecal enlargement occurred in both sexes, accompanied by soft stools in the early stages of the study, somewhat lower plasma urea concentrations and increased plasma alkaline phosphatase activity and a decreased urinary pH. This group also showed slight growth depression accompanied by transient reduction in food intake; in males the body weights remained relatively low throughout the experimental period. Furthermore, bilirubin level was increased in females and total protein level was decreased in males of the high-dose group. The above changes were considered adaptive responses or chance effects rather than manifestations of clear toxicity. Therefore the NOAEL could be set at 5% in diet (ca. 4000 mg/kg bw). The low- and intermediate-dose groups did not show any compound-related untoward effect, so it was concluded that the mid dose (1%) of about 750 mg/kg bw/d, was the NOEL.

Endpoint:
sub-chronic toxicity: oral
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
weight of evidence
Justification for type of information:
REPORTING FORMAT FOR THE ANALOGUE APPROACH
See "attached justification".
Reason / purpose:
read-across source
Specific details on test material used for the study:
TEST MATERIAL
- Name of test material: neohesperidin
- IUPAC name: 5-hydroxy-2-(3-hydroxy-4-methoxyphenyl)-4-oxo-3,4-dihydro-2H-chromen-7-yl 2-O-(6-deoxyhexopyranosyl)hexopyranoside
- Molecular formula: C28H34O15
- Molecular weight: 610.5606
- Smiles notation: COc1ccc(cc1O)C2CC(=O)c3c(O)cc(OC4OC(CO)C(O)C(O)C4OC5OC(C)C(O)C(O)C5O)cc3O2
- InChl: InChI= 1/C28H34O15/c1-10-21(33)23(35)25(37)27(39-10)43-26-24(36)22(34)19(9-29)42-28(26)40-12-6-14(31)20-15(32)8-17(41-18(20)7-12)11-3-4-16(38-2)13(30)5-11/h3-7,10,17,19,21-31,33-37H,8-9H2,1-2H3
Clinical signs:
no effects observed
Mortality:
no mortality observed
Body weight and weight changes:
effects observed, non-treatment-related
Food consumption and compound intake (if feeding study):
no effects observed
Food efficiency:
no effects observed
Water consumption and compound intake (if drinking water study):
not specified
Ophthalmological findings:
not specified
Haematological findings:
no effects observed
Clinical biochemistry findings:
not specified
Urinalysis findings:
not specified
Behaviour (functional findings):
not specified
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
no effects observed
Gross pathological findings:
no effects observed
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
no effects observed
Histopathological findings: neoplastic:
not specified
Other effects:
no effects observed
Key result
Dose descriptor:
NOAEL
Effect level:
ca. 750 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male
Basis for effect level:
other: Read-across from analogue substance.
Remarks on result:
other: For the analogue substance, no effects observed at 1% in diet; ca. 750 mg/kg/day calculated for rats with a mean body weight of ca. 250 g and ca. 20 g/day of food consumption.
Key result
Critical effects observed:
no
Conclusions:
Based on the read-across approach, the NOAEL for oral toxicity of neohesperidin in rats can be set to 750 mg/kg bw per day.
Executive summary:

A sub-chronic oral toxicity study was conducted with the analogue substance hesperidin (no guideline available, no analytical verification of doses). The study is well documented and meets generally accepted scientific principles. Six to eight healthy male albino rats per group were fed a standard diet containing the test item at doses of 0 (control), 0.0625 or 1% for 200 days. The continued feeding of the analogue substance to healthy albino rats in a standard diet for a period of 200 days, in concentrations as high as 1% of the diet by weight, gave no evidences of cumulative injury as judged by growth curves, weights of important viscera, and macroscopic and microscopic examination of these tissues. There was no difference between control rats and those receiving the test item, giving a negative result. Therefore, the NOAEL for the analogue substance can be set to 1% (ca. 750 mg) by weight per day in food. Based on the read-across approach, the NOAEL for oral toxicity of the target substance in rats can be set to 750 mg/kg bw per day.

Endpoint:
sub-chronic toxicity: oral
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
weight of evidence
Justification for type of information:
REPORTING FORMAT FOR THE ANALOGUE APPROACH
See "attached justification".
Reason / purpose:
read-across source
Specific details on test material used for the study:
TEST MATERIAL
- Name of test material: neohesperidin
- IUPAC name: 5-hydroxy-2-(3-hydroxy-4-methoxyphenyl)-4-oxo-3,4-dihydro-2H-chromen-7-yl 2-O-(6-deoxyhexopyranosyl)hexopyranoside
- Molecular formula: C28H34O15
- Molecular weight: 610.5606
- Smiles notation: COc1ccc(cc1O)C2CC(=O)c3c(O)cc(OC4OC(CO)C(O)C(O)C4OC5OC(C)C(O)C(O)C5O)cc3O2
- InChl: InChI= 1/C28H34O15/c1-10-21(33)23(35)25(37)27(39-10)43-26-24(36)22(34)19(9-29)42-28(26)40-12-6-14(31)20-15(32)8-17(41-18(20)7-12)11-3-4-16(38-2)13(30)5-11/h3-7,10,17,19,21-31,33-37H,8-9H2,1-2H3
Clinical signs:
no effects observed
Mortality:
mortality observed, non-treatment-related
Body weight and weight changes:
effects observed, non-treatment-related
Food consumption and compound intake (if feeding study):
no effects observed
Food efficiency:
no effects observed
Water consumption and compound intake (if drinking water study):
no effects observed
Ophthalmological findings:
not specified
Haematological findings:
no effects observed
Clinical biochemistry findings:
effects observed, treatment-related
Urinalysis findings:
not specified
Behaviour (functional findings):
not specified
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, non-treatment-related
Gross pathological findings:
effects observed, non-treatment-related
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
effects observed, non-treatment-related
Histopathological findings: neoplastic:
not specified
Other effects:
no effects observed
Key result
Dose descriptor:
NOAEL
Effect level:
4.88 other: % in diet
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: read-across from analogue substance.
Key result
Critical effects observed:
no
Conclusions:
Based on the read-across approach, the target substance has a sub-chronic oral NOAEL of 4.88% in the diet in B6C3F1 mice.
Executive summary:

A subchronic toxicity study was conducted for the analogue substance methyl hesperidin by a method similar to OECD 408. One hundred and twenty B6C3F1 mice (60 males and 60 females) were fed powdered diet containing 0 (control), 0.3, 0.6, 1.25, 2.5 and 5.0% test substance for 13 weeks. No significant changes were revealed in any of the measured parameters, except for the clinical chemistry: females fed 2.5% and 5.0% test substance demonstrated significant decreases in glucose level. However, the lack of any clear dose-relation or adverse effects on body weight, organ weight, gross pathological and histopathological appearance indicated an absence of toxicity. Thus, it can be concluded that methyl hesperidin exerts no toxic influence in mice of either sex at levels up to 5.0% in the diet. Based on the study results, the NOAEL for the test item was set at 5.0% in diet. Based on the read-across approach, the target substance has a sub-chronic oral NOAEL of 4.88% in the diet in B6C3F1 mice.

Endpoint:
sub-chronic toxicity: oral
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
weight of evidence
Justification for type of information:
REPORTING FORMAT FOR THE ANALOGUE APPROACH
See "attached justification".
Reason / purpose:
read-across source
Specific details on test material used for the study:
TEST MATERIAL
- Name of test material: neohesperidin
- IUPAC name: 5-hydroxy-2-(3-hydroxy-4-methoxyphenyl)-4-oxo-3,4-dihydro-2H-chromen-7-yl 2-O-(6-deoxyhexopyranosyl)hexopyranoside
- Molecular formula: C28H34O15
- Molecular weight: 610.5606
- Smiles notation: COc1ccc(cc1O)C2CC(=O)c3c(O)cc(OC4OC(CO)C(O)C(O)C4OC5OC(C)C(O)C(O)C5O)cc3O2
- InChl: InChI= 1/C28H34O15/c1-10-21(33)23(35)25(37)27(39-10)43-26-24(36)22(34)19(9-29)42-28(26)40-12-6-14(31)20-15(32)8-17(41-18(20)7-12)11-3-4-16(38-2)13(30)5-11/h3-7,10,17,19,21-31,33-37H,8-9H2,1-2H3
Clinical signs:
effects observed, non-treatment-related
Mortality:
mortality observed, non-treatment-related
Body weight and weight changes:
effects observed, non-treatment-related
Food consumption and compound intake (if feeding study):
effects observed, non-treatment-related
Water consumption and compound intake (if drinking water study):
effects observed, non-treatment-related
Ophthalmological findings:
no effects observed
Haematological findings:
no effects observed
Clinical biochemistry findings:
effects observed, treatment-related
Urinalysis findings:
effects observed, treatment-related
Behaviour (functional findings):
no effects observed
Immunological findings:
not specified
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Gross pathological findings:
no effects observed
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
no effects observed
Histopathological findings: neoplastic:
not specified
Other effects:
no effects observed
Key result
Dose descriptor:
NOEL
Effect level:
ca. 748 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: Read-across from analogue substance.
Key result
Dose descriptor:
NOAEL
Effect level:
ca. 3 987 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: Read-across from analogue substance.
Key result
Critical effects observed:
no
Conclusions:
Based on the read-across approach, the target substance has a sub-chronic oral NOAEL of ca. 4000 mg/kg bw/d in rats, and a NOEL ca. 750 mg/kg bw/d.
Executive summary:

A study on the sub-chronic toxicity of the test item was performed by a method similar to OECD 408. The test item was administered to groups of 20 male and 20 female Wistar rats at dietary levels of 0 (control), 0.2, 1.0 and 5.0% for 91 days. No treatment-related ophthalmoscopical, haematological or histopathological effects were observed. In the high-dose group, a marked caecal enlargement occurred in both sexes, accompanied by soft stools in the early stages of the study, somewhat lower plasma urea concentrations and increased plasma alkaline phosphatase activity and a decreased urinary pH. This group also showed slight growth depression accompanied by transient reduction in food intake; in males the body weights remained relatively low throughout the experimental period. Furthermore, bilirubin level was increased in females and total protein level was decreased in males of the high-dose group. The above changes were considered adaptive responses or chance effects rather than manifestations of clear toxicity. Therefore the NOAEL could be set at 5% in diet (ca. 4000 mg/kg bw). The low- and intermediate-dose groups did not show any compound-related untoward effect, so it was concluded that the mid dose (1%) of about 750 mg/kg bw/d, was the NOEL. Based on the read-across approach, the target substance has a sub-chronic oral NOAEL of ca. 4000 mg/kg bw/d in rats.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
750 mg/kg bw/day
Study duration:
subchronic
Species:
rat
Quality of whole database:
All studies avaliable have a Klimisch score of 2.

Repeated dose toxicity: inhalation - systemic effects

Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: inhalation - local effects

Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: dermal - systemic effects

Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: dermal - local effects

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

Weight of evidence: The substance is considered to be non-toxic, with an oral sub-chronic NOAEL of ca. 750 mg/kg bw/d (worst-case), based on the available information from supporting analogue substances, which are similar in structure to the target substance and share common metabolic pathways:

- Read-across from supporting substance (structural analogue or surrogate). Source: Key study. Method similar to OECD 408. The analogue substance was administered to groups of 20 male and 20 female Wistar rats at dietary levels of 0 (control), 0.2, 1.0 and 5.0% for 91 days. No significant treatment-related effects were observed at any dose; no effects were observed at the mid and low doses. The NOAEL of the analogue substance neohesperidin dihydrochalcone for oral sub-chronic toxicity in rats was found to be ca. 4000 mg/kg bw/d (5% in diet) and the NOEL ca. 750 mg/kg bw (1%). Based on the read-across approach, the NOAEL of the target substance is ca. 3987 mg/kg bw/d.

-Read-across from supporting substance (structural analogue or surrogate). Source: Study well documented, meets generally accepted scientific criteria (no guideline available). Six to eight healthy male albino rats per group were fed a standard diet containing the test item at doses of 0 (control), 0.0625 or 1% for 200 days. No differences were observed between control rats and those receiving the test item. Therefore, the NOAEL for the analogue substance was set to 1% (ca. 750 mg) by weight per day in food. Based on the read-across approach, the NOAEL for oral toxicity of neohesperidin in rats can be set to 750 mg/kg bw per day.

-Read-across from supporting substance (structural analogue or surrogate). Source: Method similar to OECD 408. One hundred and twenty B6C3F1 mice (60 males and 60 females) were fed powdered diet containing 0 (control), 0.3, 0.6, 1.25, 2.5 and 5.0% test substance for 13 weeks. No significant changes were revealed in any of the measured parameters, except for the clinical chemistry; the lack of any clear dose-relation or adverse effects on body weight, organ weight, gross pathological and histopathological appearance indicated an absence of toxicity. The NOAEL of the analogue substance methyl hesperidin for oral sub-chronic toxicity in mice was found to be 5% in diet. Based on the read-across approach, the NOAEL of the target substance is 4.88% in diet.

Justification for classification or non-classification

Based on the available information (NOAEL ca. 750 mg/kg bw/d in rats), the substance is not classified for repeated dose toxicity (STOT RE) in accordance with CLP Regulation (EU) No. 1272/2008.